DOCK8-expressing T follicular helper cells newly generated beyond self-organized criticality cause systemic lupus erythematosus.

Pathogens including autoantigens all failed to induce systemic lupus erythematosus (SLE). We, instead, studied the integrity of host's immune response that recognized pathogen. By stimulating TCR with an antigen repeatedly to levels that surpass host's steady-state response, self-organized criticality, SLE was induced in mice normally not prone to autoimmunity, wherein T follicular helper (Tfh) cells expressing the guanine nucleotide exchange factor DOCK8 on the cell surface were newly generated. DOCK8+Tfh cells passed through TCR re-revision and induced varieties of autoantibody and lupus lesions. They existed in splenic red pulp and peripheral blood of active lupus patients, which subsequently declined after therapy. Autoantibodies and disease were healed by anti-DOCK8 antibody in the mice including SLE-model (NZBxNZW) F1 mice. Thus, DOCK8+Tfh cells generated after repeated TCR stimulation by immunogenic form of pathogen, either exogenous or endogenous, in combination with HLA to levels that surpass system's self-organized criticality, cause SLE.

FBP17-mediated finger-like membrane protrusions in cell competition between normal and RasV12-transformed cells.

At the initial stage of carcinogenesis, cell competition often occurs between newly emerging transformed cells and the neighboring normal cells, leading to the elimination of transformed cells from the epithelial layer. For instance, when RasV12-transformed cells are surrounded by normal cells, RasV12 cells are apically extruded from the epithelium. However, the underlying mechanisms of this tumor-suppressive process still remain enigmatic. We first show by electron microscopic analysis that characteristic finger-like membrane protrusions are projected from both normal and RasV12 cells at their interface. In addition, FBP17, a member of the F-BAR proteins, accumulates in RasV12 cells, as well as surrounding normal cells, which plays a positive role in the formation of finger-like protrusions and apical elimination of RasV12 cells. Furthermore, cdc42 acts upstream of these processes. These results suggest that the cdc42/FBP17 pathway is a crucial trigger of cell competition, inducing "protrusion to protrusion response" between normal and RasV12-transformed cells.

Conditional Upregulation of IFN-α Alone Is Sufficient to Induce Systemic Lupus Erythematosus.

The cause of systemic lupus erythematosus (SLE) is unknown. IFN-α has been suggested as a causative agent of SLE; however, it was not proven, and to what extent and how IFN-α contributes to the disease is unknown. We studied the contribution of IFN-α to SLE by generating inducible IFN-α transgenic mice and directly show that conditional upregulation of IFN-α alone induces a typical manifestation of SLE in the mice not prone to autoimmunity, such as serum immune complex, autoantibody against dsDNA (anti-dsDNA Ab), and the organ manifestations classical to SLE, such as immune complex-deposited glomerulonephritis, classical splenic onion-skin lesion, alopecia, epidermal liquefaction, and positive lupus band test of the skin. In the spleen of mice, activated effector CD4 T cells, IFN-γ-producing CD8 T cells, B220+CD86+ cells, and CD11c+CD86+ cells were increased, and the T cells produced increased amounts of IL-4, IL-6, IL-17, and IFN-γ and decreased IL-2. In particular, activated CD3+CD4-CD8- double-negative T cells positive for TCRαß, B220, CD1d-teteramer, PD-1, and Helios (that produced increased amounts of IFN-γ, IL-4, IL-17, and TNF-α) were significantly expanded. They infiltrated into kidney and induced de novo glomerulonephritis and alopecia when transferred into naive recipients. Thus, sole upregulation of IFN-α is sufficient to induce SLE, and the double-negative T cells expanded by IFN-α are directly responsible for the organ manifestations, such as lupus skin disease or nephritis.

Suppression of autophagic activity by Rubicon is a signature of aging.

Autophagy, an evolutionarily conserved cytoplasmic degradation system, has been implicated as a convergent mechanism in various longevity pathways. Autophagic activity decreases with age in several organisms, but the underlying mechanism is unclear. Here, we show that the expression of Rubicon, a negative regulator of autophagy, increases in aged worm, fly and mouse tissues at transcript and/or protein levels, suggesting that an age-dependent increase in Rubicon impairs autophagy over time, and thereby curtails animal healthspan. Consistent with this idea, knockdown of Rubicon extends worm and fly lifespan and ameliorates several age-associated phenotypes. Tissue-specific experiments reveal that Rubicon knockdown in neurons has the greatest effect on lifespan. Rubicon knockout mice exhibits reductions in interstitial fibrosis in kidney and reduced α-synuclein accumulation in the brain. Rubicon is suppressed in several long-lived worms and calorie restricted mice. Taken together, our results suggest that suppression of autophagic activity by Rubicon is one of signatures of aging.

CD133 Modulate HIF-1α Expression under Hypoxia in EMT Phenotype Pancreatic Cancer Stem-Like Cells.

UNLABELLED: Although CD133 is a known representative cancer stem cell marker, its function in tumor aggressiveness under hypoxia is not fully known. The aim of this study is to demonstrate that CD133 regulates hypoxia inducible factor (HIF)-1α expression with tumor migration. The CD133⁺ pancreatic cancer cell line, Capan1M9, was compared with the CD133(-) cell line, shCD133M9, under hypoxia. HIF-1α expression levels were compared by Western blot, HIF-1α nucleus translocation assay and real-time (RT)-PCR. The hypoxia responsive element (HRE) was observed by luciferase assay. The migration ability was analyzed by migration and wound healing assays. Epithelial mesenchymal transition (EMT) related genes were analyzed by real-time RT-PCR. HIF-1α was highly expressed in Capan1M9 compared to shCD133M9 under hypoxia because of the high activation of HRE. Furthermore, the migration ability of Capan1M9 was higher than that of shCD133M9 under hypoxia, suggesting higher expression of EMT related genes in Capan1M9 compared to shCD133M9. CONCLUSION: HIF-1α expression under hypoxia in CD133⁺ pancreatic cancer cells correlated with tumor cell migration through EMT gene expression. Understanding the function of CD133 in cancer aggressiveness provides a novel therapeutic approach to eradicate pancreatic cancer stem cells.

Efficient elimination of pancreatic cancer stem cells by hedgehog/GLI inhibitor GANT61 in combination with mTOR inhibition.

BACKGROUND: Pancreatic cancer is one of the most lethal malignancies. The innovative treatments are required and now the cancer stem cells (CSCs) are expected to be an effective target for novel therapies. Therefore we investigated the significance of hedgehog (Hh) signaling in the maintenance of CSC-like properties of pancreatic cancer cells, in order to discover the key molecules controlling their unique properties. METHODS: Human pancreatic cancer cell lines, Capan-1, PANC-1, MIA PaCa-2 and Capan-1 M9 were used for our experiments in DMEM/F12 medium containing 10 % fetal bovine serum. Sphere formation assay, immunofluorescence staining, flow cytometric analysis and MTT cell viability assay were performed to investigate molecular signals and the efficacy in the treatment of pancreatic cancer cells. RESULTS: Inhibition of the Hh pathway significantly reduced the expression of stem cell marker CD133 and sphere formation, an index of self-renewal capacity, demonstrating the suppression of CSC-like properties. Moreover, the GLI inhibitor GANT61 induced greater reduction in sphere formation and cell viability of pancreatic cancer cells than the smoothened (SMO) inhibitor cyclopamine. This suggests that GLI transcription factors, but not SMO membrane protein, are the key molecules in the Hh pathway. The treatment using GANT61 in combination with the inhibition of mTOR, which is another key molecule in pancreatic CSCs, resulted in the efficient reduction of cell viability and sphere formation of an inhibitor-resistant cell line, showing the strong efficacy and wide range applicability to pancreatic CSC-like cells. CONCLUSIONS: Thus, this novel combination treatment could be useful for the control of pancreatic cancer by targeting pancreatic CSCs. This is the first report of the efficient elimination of pancreatic cancer stem-like cells by the double blockage of Hh/GLI and mTOR signaling.

miR-30 family promotes migratory and invasive abilities in CD133(+) pancreatic cancer stem-like cells.

Pancreatic cancer is a deadly disease with a poor prognosis. Recently, miRNAs have been reported to be abnormally expressed in several cancers and play a role in cancer development and progression. However, the role of miRNA in cancer stem cells remains unclear. Therefore, our aim was to investigate the role of miRNA in the CD133(+) pancreatic cancer cell line Capan-1M9 because CD133 is a putative marker of pancreatic cancer stem cells. Using miRNA microarray, we found that the expression level of the miR-30 family decreased in CD133 genetic knockdown shCD133 Capan-1M9 cells. We focused on miR-30a, -30b, and -30c in the miR-30 family and created pancreatic cancer cell sublines, each transfected with these miRNAs. High expression of miR-30a, -30b, or -30c had no effect on cell proliferation and sphere forming. In contrast, these sublines were resistant to gemcitabine, which is a standard anticancer drug for pancreatic cancer, and in addition, promoted migration and invasion. Moreover, mesenchymal markers were up-regulated by these miRNAs, suggesting that mesenchymal phenotype is associated with an increase in migration and invasion. Thus, our study demonstrated that high expression of the miR-30 family modulated by CD133 promotes migratory and invasive abilities in CD133(+) pancreatic cancer cells. These findings suggest that targeted therapies to the miR-30 family contribute to the development of novel therapies for CD133(+) pancreatic cancer stem cells.

Slug contributes to gemcitabine resistance through epithelial-mesenchymal transition in CD133(+) pancreatic cancer cells.

CD133-positive pancreatic cancer is correlated with unfavorable survival despite current development of therapy. Slug acts as a master regulator of epithelial-mesenchymal transition (EMT) which is the essential process in cancer progression. The aim of this study was to investigate the role of Slug in gemcitabine treatment for CD133-positive pancreatic cancer cells. We used a previously established pancreatic cancer cell line expressing high level of CD133 (Capan-1M9), which also expresses high level of Slug. We generated Slug knock-down subclone (shSlug M9) from this cell line, and compared expression of EMT-related genes, migration, invasion and gemcitabine resistance between two cell lines. Slug knock-down in CD133-positive pancreatic cancer cell line led to the reduction of migration and invasion ability. Furthermore, Slug knock-down sensitized CD133-positive pancreatic cancer cell line to gemcitabine. These results suggest that Slug plays an important role in not only invasion ability through EMT but also gemcitabine resistance of CD133-positive pancreatic cancer cells.

In vivo cell transfer assay to detect autoreactive T cell subsets.

Among the methods used in molecular biology, in vitro biochemical assays are more common, whereas in vivo assays, including the use of animal models, are less widely employed. In our studies on systemic lupus erythematosus (SLE), we have identified a novel T cell subtype termed "autoantibody-inducing CD4 T cells" (aiCD4 T cell) that is responsible for the development of autoimmunity. In order to identify and isolate these cells, we developed a new technique that involves the transfer of candidate T cell subpopulations into naïve mice and assaying for the development of autoantibodies in the recipient mice. We have previously described an experimental system in which mice not normally prone to autoimmune diseases can be induced to develop experimental SLE. In this experimental system, autoantibody-inducing CD4 T (aiCD4 T) cells are generated via de novo T cell receptor revision at peripheral lymphoid organs. These aiCD4 T cells not only induce a variety of autoantibodies but also promote the final differentiation of CD8 T cells into cytotoxic T lymphocytes, resulting in a pathology identical to SLE. We needed to develop a new methodology to isolate this subpopulation of aiCD4 T cells. Here we describe an in vivo assay to detect and isolate aiCD4 T cells by transferring the candidate cells into naïve recipient mice and monitoring the production of the appropriate antibody or cytokine.

CD133 facilitates epithelial-mesenchymal transition through interaction with the ERK pathway in pancreatic cancer metastasis.

BACKGROUND: Pancreatic cancer is a lethal disease due to the high incidence of metastasis at the time of detection. CD133 expression in clinical pancreatic cancer correlates with poor prognosis and metastasis. However, the molecular mechanism of CD133-regulated metastasis remains unclear. In recent years, epithelial-mesenchymal transition (EMT) has been linked to cancer invasion and metastasis. In the present study we investigated the role of CD133 in pancreatic cancer metastasis and its potential regulatory network. METHODS: A highly migratory pancreatic cancer cell line, Capan1M9, was established previously. After shRNA was stable transducted to knock down CD133 in Capan1M9 cells, gene expression was profiled by DNA microarray. Orthotopic, splenic and intravenous transplantation mouse models were set up to examine the tumorigenesis and metastatic capabilities of these cells. In further experiments, real-time RT-PCR, Western blot and co-immunoprecipitate were conducted to evaluate the interactions of CD133, Slug, N-cadherin, ERK1/2 and SRC. RESULTS: We found that CD133+ human pancreatic cancer cells were prone to generating metastatic nodules in in vivo models using immunodeficient mice. In contrast, CD133 knockdown suppressed cancer invasion and metastasis in vivo. Gene profiling analysis suggested that CD133 modulated mesenchymal characteristics including the expression of EMT-related genes, such as Slug and N-cadherin. These genes were down-regulated following CD133 knockdown. Moreover, CD133 expression could be modulated by the extracellular signal-regulated kinase (ERK)1/2 and SRC signaling pathways. The binding of CD133 to ERK1/2 and SRC acts as an indispensable mediator of N-cadherin expression. CONCLUSIONS: These results demonstrate that CD133 plays a critical role in facilitating the EMT regulatory loop, specifically by upregulating N-cadherin expression, leading to the invasion and metastasis of pancreatic cancer cells. Our study provides a novel insight into the function of CD133 in the EMT program and a better understanding of the mechanism underlying the involvement of CD133 in pancreatic cancer metastasis.

mTOR plays critical roles in pancreatic cancer stem cells through specific and stemness-related functions.

Pancreatic cancer is characterized by near-universal mutations in KRAS. The mammalian target of rapamycin (mTOR), which functions downstream of RAS, has divergent effects on stem cells. In the present study, we investigated the significance of the mTOR pathway in maintaining the properties of pancreatic cancer stem cells. The mTOR inhibitor, rapamycin, reduced the viability of CD133(+) pancreatic cancer cells and sphere formation which is an index of self-renewal of stem-like cells, indicating that the mTOR pathway functions to maintain cancer stem-like cells. Further, rapamycin had different effects on CD133(+) cells compared to cyclopamine which is an inhibitor of the Hedgehog pathway. Thus, the mTOR pathway has a distinct role although both pathways maintain pancreatic cancer stem cells. Therefore, mTOR might be a promising target to eliminate pancreatic cancer stem cells.

Expansion of PD-1-positive effector CD4 T cells in an experimental model of SLE: contribution to the self-organized criticality theory.

We have developed a systems biology concept to explain the origin of systemic autoimmunity. From our studies of systemic lupus erythematosus (SLE) we have concluded that this disease is the inevitable consequence of over-stimulating the host's immune system by repeated exposure to antigen to levels that surpass a critical threshold, which we term the system's "self-organized criticality". We observed that overstimulation of CD4 T cells in mice led to the development of autoantibody-inducing CD4 T cells (aiCD4 T) capable of generating various autoantibodies and pathological lesions identical to those observed in SLE. We show here that this is accompanied by the significant expansion of a novel population of effector T cells characterized by expression of programmed death-1 (PD-1)-positive, CD27(low), CD127(low), CCR7(low) and CD44(high)CD62L(low) markers, as well as increased production of IL-2 and IL-6. In addition, repeated immunization caused the expansion of CD8 T cells into fully-matured cytotoxic T lymphocytes (CTL) that express Ly6C(high)CD122(high) effector and memory markers. Thus, overstimulation with antigen leads to the expansion of a novel effector CD4 T cell population that expresses an unusual memory marker, PD-1, and that may contribute to the pathogenesis of SLE.

Interferon-alpha modulates the chemosensitivity of CD133-expressing pancreatic cancer cells to gemcitabine.

Pancreatic cancer is a lethal disease as current chemotherapies with gemcitabine (GEM) are still insufficient. Accumulating evidence suggests that cancer stem cells (CSC) are responsible for chemoresistance and that CD133 is one of the CSC markers in pancreatic cancer. Interferon-alpha (IFN-α), a cytokine with pleiotropic effects, has direct cytotoxic and cytostatic effects on tumor cells. The aim of the present study was to investigate whether IFN-α can modulate the chemosensitivity of a human pancreatic cancer cell line, Capan-1, to GEM. Cell cycles were evaluated for response to GEM with and without IFN-α by BrdU assay. GEM inhibited Capan-1 cell growth in a dose-dependent manner. GEM (IC(50); 100 ng/mL) treatment reduced the number of both CD133(+) and CD133(-) cells in the S phase, induced apoptosis of CD133(-) cells more than that of CD133(+) cells and increased accumulation of CD133(+) cells into the G0/G1 phase. These results infer that CD133(+) cells take shelter into the G0/G1 phase from GEM treatment. IFN-α modulated CD133(+) cells from the G0/G1 phase to the S phase. Consequently, apoptosis was accelerated in both CD133(+) and CD133(-) cells after IFN-α combined with GEM treatment. Furthermore, GEM combined with IFN-α treatment showed a significant tumor suppressive effect in the in vivo study. Importantly, CD133(+) cells showed CSC-like properties, such as generation of spheres, highly invasive ability and high tumorigenesis. These results suggest that IFN-α, as a modulator, could contribute to the treatment of CD133(+) cancer cells and be effective in combined chemotherapies with GEM for pancreatic cancer stem-like cells.

Establishment of a highly migratory subclone reveals that CD133 contributes to migration and invasion through epithelial-mesenchymal transition in pancreatic cancer.

Pancreatic cancer is a lethal disease because of invasion and early metastasis. Although CD133, a marker of cancer stem cells (CSCs) in a variety of solid tumors, has been studied in recent decades, its function remains obscure. Recent reports suggest that epithelial-mesenchymal transition (EMT) may be related to the properties of CSCs. In this study, we investigated whether CSC markers are associated with EMT. For Capan1M9, a highly migratory cell subclone established from human pancreatic cancer cell line Capan-1, CD133 expression, migration, and invasion were greater than for the parent cells. In Capan1M9 cells, the EMT-related transcription factors Slug and Snail were up-regulated, and N-cadherin and fibronectin were also substantially increased. In contrast, occludin and desmoplakin were suppressed. Knockdown of endogenous CD133 in the Capan1M9 cells led to Slug suppression and reduction of migration and invasion. Taken together, CD133 has an important role in migration and invasion by facilitating EMT in pancreatic cancer cells.

Self-organized criticality theory of autoimmunity.

BACKGROUND: The cause of autoimmunity, which is unknown, is investigated from a different angle, i.e., the defect in immune 'system', to explain the cause of autoimmunity. METHODOLOGY/PRINCIPAL FINDINGS: Repeated immunization with antigen causes systemic autoimmunity in mice otherwise not prone to spontaneous autoimmune diseases. Overstimulation of CD4(+) T cells led to the development of autoantibody-inducing CD4(+) T (aiCD4(+) T) cell which had undergone T cell receptor (TCR) revision and was capable of inducing autoantibodies. The aiCD4(+) T cell was induced by de novo TCR revision but not by cross-reaction, and subsequently overstimulated CD8(+) T cells, driving them to become antigen-specific cytotoxic T lymphocytes (CTL). These CTLs could be further matured by antigen cross-presentation, after which they caused autoimmune tissue injury akin to systemic lupus erythematosus (SLE). CONCLUSIONS/SIGNIFICANCE: Systemic autoimmunity appears to be the inevitable consequence of over-stimulating the host's immune 'system' by repeated immunization with antigen, to the levels that surpass system's self-organized criticality.